The objective of the proposed research is to develop a revolutionary DNA sequencing technology that is based on pulling DNA strands off a solid surface when stretched under an electric field. It will replace the electrophoresis-based technologies by a separation-at-a-stretch method to separate DNA strands in different lengths produced from the Sanger reaction. Separation is accomplished by attaching the DNA strands to a solid surface and stretching them under an electric field. The anchor that links the DNA to the surface is designed so that the critical force that is needed to detach the DNA strands from the surface is independent of the DNA length, while the stretching force that is applied to the DNA is proportional to the DNA length. Therefore, as the strength of the electric field gradually increases, longer DNA strands are detached first. Through a fluorescence resonance energy transfer (FRET) mechanism, detachment of the DNA strand can be directly detected from the fluorescence signals, which can be used to determine the sequence of the template DNA. Significantly, this simple and efficient method, in principle, has no upper limit on the length of the DNA strands that can be separated based on their length. We propose to use this technology to increase both the read length and the read speed of the current Sanger cycling sequencing process, while eliminating the need of costly equipments for capillary array electrophoresis, thereby reducing the current cost of DNA sequencing by 3 to 4 orders of magnitude. In addition, we propose to use this technology to separate chromosome-sized DNA molecules whose lengths exceed the current limit of electrophoresis-based technologies, with the goal of developing a promising tool to prepare samples for new DNA sequencing approaches (such as nanopore sequencing and sequencing-by-extension) where very long DNA molecules (more than several hundred million base pairs) are needed. In our preliminary studies, we have been able to achieve single-base resolution on the position of adenine (A) base in a fragment of p53 gene determined by stretching DNA strands (with 410-430 bases) produced from ddATP-terminated reaction. We have also demonstrated efficient separation of lambda dsDNA (48,502 bp) from human genomic dsDNA (100 kbp?1 Mbp) based on their length difference, using the proposed method. Further development of this technology as proposed here includes: (1) determination of the DNA sequence by combining data obtained from stretching DNA strands produced from all four dideoxy (ddATP, ddTTP, ddCTP, and ddGTP)-terminated reactions, (2) evaluation of the phred quality value as a function of base position and the read length of this sequencing technology, (3) investigating how the process parameters affect the phred quality value and the read length, thereby searching for optimized process parameters to improve the performance of the technology, (4) increasing the read speed of this method by programming the voltage source and the fluorescence signal detection equipment, and (5) separation of chromosome-sized DNA molecules (10 million to several billion base pairs) based on their lengths. PROJECT HEALTH RELEVANCE A revolutionary DNA sequencing technology will be developed based on stretching DNA molecules attached to a surface under the influence of an electric field. Successful development of the technology will reduce the cost of sequencing a mammalian-sized genome by 3 to 4 orders of magnitude, which will enable the use of comprehensive genomic sequence information in the study of biology and disease.